High sensitivity assay for detection of nucleoside diphosphate production

Abstract

The present invention provides assays for detecting ADP, GDP and inorganic phosphate. These assays can be used directly to detect the presence of ADP, GDP and inorganic phosphate or can be used as part of a number of methods for identifying candidate agents that bind to a target protein or serve as modulators of the biological activity of a target protein.

1. A method for identifying a modulator of troponin activity, comprising the steps of:
 (a) adding a candidate agent to a mixture comprising troponin, tropomyosin, myosin, and actin under conditions that normally allow production of ADP; 
 (b) determining the ADP level or production rate in the mixture of step (a); and 
 (c) comparing the ADP level or production rate from step (b) with a control value from an identical mixture but without the candidate agent, wherein an increase or decrease in the ADP level or production rate from step (b) when compared to the control value allows identification of the candidate agent as a modulator of troponin activity. 
 
     
     
       2. A method for identifying a modulator of troponin activity, comprising the steps of:
 (a) adding a candidate agent to a mixture comprising a troponin-tropomyosin complex, myosin, and actin under conditions that normally allow production of ADP; 
 (b) determining the ADP level or production rate in the mixture of step (a); and 
 (c) comparing the ADP level or production rate from step (b) with a control value from an identical mixture but without the candidate agent, wherein an increase or decrease in the ADP level or production rate from step (b) when compared to the control value allows identification of the candidate agent as a modulator of troponin activity. 
 
     
     
       3. The method of  claim 1  or  2 , wherein an increase in the ADP level or production rate from step (b) when compared to the control value allows identification of the candidate agent as an agonist of troponin activity. 
     
     
       4. The method of  claim 1  or  2 , wherein a decrease in the ADP level or production rate from step (b) when compared to the control value allows identification of the candidate agent as an antagonist of troponin activity. 
     
     
       5. The method of  claim 1  or  2 , wherein the mixture in (a) further comprises ATP. 
     
     
       6. The method of  claim 1  or  2 , wherein step (b) comprises an enzymatic reaction that utilizes ADP as a substrate under conditions normally allow ADP to be utilized as a substrate. 
     
     
       7. The method of  claim 6 , wherein the enzymatic reaction results in formation of a detectable compound. 
     
     
       8. The method of  claim 6 , wherein the enzymatic reaction results in formation of pyruvate or hydrogen peroxide. 
     
     
       9. The method of  claim 1  or  2 , wherein step (a) is performed in plurality with each of a plurality of candidate agents added to one of a plurality of identical mixtures. 
     
     
       10. The method of  claim 1  or  2 , wherein step (a) is performed in plurality with the candidate agent each at a distinct concentration added to one of a plurality of identical mixtures. 
     
     
       11. The method of  claim 9 , wherein step (a) is performed automatically. 
     
     
       12. The method of  claim 9 , wherein steps (a)-(c) are performed automatically. 
     
     
       13. The method of  claim 10 , wherein step (a) is performed automatically. 
     
     
       14. The method of  claim 10 , wherein steps (a)-(c) are performed automatically. 
     
     
       15. The method of  claim 1  or  2 , further comprising a step of detecting binding between the candidate agent and troponin, wherein the presence of binding between the candidate agent and troponin indicates the candidate agent as a modulator of troponin activity.